Transposable genetic elements constitute a diverse group of discrete DNA segments with a capability of moving within and between genomes (1). They are abundant in all kingdoms of life and present in virtually every genome examined to date (1, 2). A wealth of data from sequenced genomes has implicated the fundamental importance of mobile DNA in shaping genomes during evolution (3-6). The increasing knowledge of DNA mobility mechanisms has facilitated the versatile use of transposable elements for research purposes and provided efficient tools for a variety of applications including genome-wide insertional mutagenesis, protein engineering, transgenesis, and gene therapy (7-9).
Phage Mu is a mobile DNA element encoding MuA transposase, the critical catalytic component of the mobilization machinery. MuA transposase belongs to the retroviral integrase superfamily of proteins. It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA into non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. It would be advantageous if the primary component of the Mu transpososome, the MuA transposase, could be modified for better performance with regard the applications. Here, we have mutated MuA protein using random mutagenesis methods in order to identify MuA variants with enhanced transpositional activity. Initially, we generated a pool of randomly mutated MuA-expressing plasmid clones, and by screening approx. 60.000 clones identified several hundred clones expressing hyperactive MuA variants. The identification employed a genetic screen using a quantitative in vivo transposition assay. This quantitative assay is based on the mobilization of a reporter transposon inside Escherichia coli cells. In this assay, individual transposition events are scored as blue microcolonies (papillae) growing on otherwise whitish bacterial colonies. The mutant-phenotype-causing nucleotide changes were then identified by DNA sequencing for 92 MuA-variant-expressing clones. Subsequently, the identified nucleotide changes, translated as amino acid changes, were mapped onto the primary amino acid sequence of MuA transposase. A total of 47 changes were selected for further scrutiny. Corresponding amino acid changes were introduced individually into MuA. These single-amino-acid-change MuA variants were then analyzed by the papillation analysis for their transpositional activity. This way, we identified 33 single-substitution MuA variants, which generated more than 2-fold excess papillae in the assay relative to the wild type MuA. We further showed that enhanced transpositional activities identified in the in vivo papillation assay were largely recapitulated in two other assays relevant for MuA-based transposon applications, namely: (i) introduction of transpososomes into bacterial cells for genomic integration and (ii) generation of recombinant molecules by in vitro transposition. These assays were performed using purified proteins expressed in Escherichia coli. In addition, we showed that by combining two or three activity-enhancing amino acid changes, cumulative enhancement in the protein activity could be attained. Thus, by combining several beneficial amino acid changes within a single MuA polypeptide, it is possible to generate MuA transposase variants that possess a substantially enhanced transpositional activity.
Many mobile DNA elements transpose via a DNA intermediate. This group of elements includes bacterial and eukaryotic transposons as well as transposing bacteriophages such as phage Mu. This phage utilizes DNA transposition as an important step in its propagation cycle. Owing to its efficient DNA mobilization capacity in vivo (10) and the early development of an in vitro system (11), phage Mu has served as an important model system for DNA transposition studies in general (12). Mu encodes MuA transposase, which catalyzes the critical steps of transposition: (i) initial cleavages at the transposon-host boundaries (donor cleavage) and (ii) covalent integration of the transposon into the target DNA (strand transfer). These steps proceed via sequential structural transitions within a nucleoprotein complex, a transpososome (12-16), the core of which contains four MuA molecules and two synapsed transposon ends (17,18). In vivo, the critical MuA-catalyzed reaction steps also involve the phage-encoded MuB targeting protein, host-encoded DNA architectural proteins (HU and IHF), certain DNA cofactors (MuA binding sites and transpositional enhancer sequence), as well as stringent DNA topology (19). The critical reaction steps mimicking Mu transposition into external target DNA can be reconstituted in vitro using MuA transposase, 50 bp Mu R-end DNA segments, and target DNA as the only macromolecular components (18, 20). Such a minimal system has been instrumental for the detailed analyses on the molecular mechanisms of Mu transposition (21-23). A versatile use of the reaction series with custom-designed substrates has generated a wealth of tools for molecular biology applications (24-29) and produced novel strategies for genetics/genomics research (30-34).
MuA is a 75-kDa protein (663 amino acids) and can be divided into structurally and functionally defined major domains (I, II, III) and subdomains (Iα, Iβ, Iγ; IIα, IIβ; IIIα, IIIβ) (35-39) (see also FIG. 1). The N-terminal subdomain Iα promotes transpososome assembly via an initial binding to a specific transpositional enhancer sequence (40, 41). The specific DNA binding to transposon ends, crucial for the transpososome assembly, is mediated through amino acid residues located in subdomains Iβ and Iγ (37,38). Subdomain IIα contains the critical DDE-motif of acidic residues (D269, D336 and E392), which is involved in the metal ion coordination during the catalysis (42, 43). Subdomains IIβ and IIα participate in nonspecific DNA binding, and they appear important during structural transitions (17,43). Subdomain IIIα also displays a cryptic endonuclease activity, which is required for the removal of the attached host DNA following the integration of infecting Mu (44, 45). The C-terminal subdomain IIIβ is responsible for the interaction with the phage-encoded MuB protein, important in targeting transposition into distal target sites (46-49). This subdomain is also important in interacting with the host-encoded ClpX protein, a factor which remodels the transpososome for disassembly (50). While all MuA subdomains are required for efficient phage Mu transposition inside Escherichia coli, the terminal subdomains Iα and IIIβ become dispensable in certain in vivo and in vitro conditions with appropriately altered DNA substrates and/or suitably modified reaction milieu (51,52).
Here, we have employed random mutagenesis to generate substitutions in MuA. From thousands of MuA substitution variants, we screened hyperactive transposase mutants and identified amino acid changes responsible for the observed phenotypic change. A combination of several hyperactivity-causing changes had a cumulative effect on the protein activity.